Nitric oxide-induced hyperpolarization stimulates low-conductance Na⁺ channel of rat CCD

We used the patch-clamp technique in the split-open cortical collecting duct (CCD) to investigate the effect of nitric oxide (NO) on the low-conductance (6-pS) Na⁺ channel that can be blocked by 1 μM amiloride. We confirmed that the number of Na⁺ channels increased significantly in CCDs of rats on a low-Na⁺ diet (17). Application of 100 μM N^G-nitro-L-arginine methyl ester (L-NAME), an agent that blocks endogenous NO synthase, reduced NP₀ [the product of channel number (N) and open probability (P₀)] to 45% of the control value. The effect of L-NAME was specific, since addition of D-NAME, which does not inhibit NO synthase, did not change the activity of the Na⁺ channel. That the effect of L-NAME results from inhibition of NO synthase is further confirmed by experiments in which addition of an exogenous NO donor, either 10 uM S-nitroso-N-acetyl penicillamine or sodium nitroprusside (SNP), restored the Na⁺ channel activity when it had been blocked by L-NAME. The action of NO involves a guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent pathway, since 100 μM 8-bromo-cGMP (8-BrcGMP) mimicked the effect of SNAP on K⁺ channels. However, 100 μM 8-BrcGMP did not alter the activity of Na⁺ channels in inside-out patches, suggesting an indirect action. Because the Na⁺ channel is activated by hyperpolarization (19) and NO stimulates basolateral K⁺ channels (16), we tested whether hyperpolarization mediated the effect of NO. In perforated whole cell recordings, addition of L-NAME depolarized the cell membrane from -73 to -51 mV, and application of 10 uM SNP repolarized the membrane to -68 mV. Furthermore, the L-NAME-induced decrease in NP₀ was effectively restored by -25 mV hyperpolarization of the patch membranes, and addition of 2 mM Ba²⁺ also abolished the effect of L-NAME. We concluded that the stimulatory effect of NO on the Na⁺ channel is an indirect effect mediated by a NO-induced increase of basolateral K⁺ conductance.